Cellular and molecular events controlling acquisition of cytotoxic activity by melanoma-reactive CD4⁺ T cells in vivo

While there is an abundance of studies on cytotoxic CD8⁺ T cells in cancer immunotherapy, CD4⁺ T cells with cytotoxic potential are receiving increasing attention from the scientific community. Previously, our lab has underscored the significance of tumour reactive CD4⁺ T cells which acquire cytotoxic activity during immunotherapy of malignant melanoma. This study aims to analyse and characterise the molecular and cellular mechanisms underlying the function of these cytotoxic CD4⁺ T cells. On protein and transcript level, cytotoxic tumour infiltrating CD4⁺Trp1 T cells exhibited a highly plastic phenotype: Th1 and Th2 specific transcription factors Gata3 and T-bet were co-expressed and inflammatory cytokines IFNγ, TNF-α and IL-2 were secreted. Additionally, CD8⁺ lineage specific transcription factor Runx3 expression was elevated and correlated highly with GzmB expression. However, and in contrast to classical CD8⁺ CTLs, cytotoxic CD4⁺Trp1 T cells lacked expression of CD8⁺ transcription factor Eomes. In depth microarray analysis via Canonical Correspondence Analysis (CCAM) revealed a high correlation of tumour infiltrating CD4⁺Trp1 cells with a full effector CD8⁺ T cell gene signature rather than a CD4⁺ or CD8⁺ memory phenotype. The strong correspondence with differentiated CD8⁺ effector T cells prompted the investigation of the role of mTOR signalling in CD4⁺ cytotoxicity as mTOR activity is crucial for CD8⁺ effector differentiation. Inhibition of mTORC1 activity by administration of rapamycin and genetic engineering of CD4⁺Trp1 cells was evaluated. Disruption of mTORC1 signalling counteracted the acquisition and/or maintenance of a cytotoxic phenotype whilst preserving the capacity to produce inflammatory cytokines. This study illustrates the complexity of this highly plastic, cytotoxic CD4⁺ T cell subset and highlights the importance of mTORC1 signalling for the cytotoxic activity of tumour specific CD4⁺Trp1 T cells

The PERK Inhibitor GSK2606414 Enhances Reovirus Infection in Head and Neck Squamous Cell Carcinoma via an ATF4-Dependent Mechanism.

Reovirus type 3 Dearing (reovirus) is a tumor-selective oncolytic virus currently under evaluation in clinical trials. Here, we report that the therapeutic efficacy of reovirus in head and neck squamous cell cancer can be enhanced by targeting the unfolded protein response (UPR) kinase, protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). PERK inhibition by GSK2606414 increased reovirus efficacy in both 2D and 3D models in vitro, while perturbing the normal host cell response to reovirus-induced endoplasmic reticulum (ER) stress. UPR reporter constructs were used for live-cell 3D spheroid imaging. Profiling of eIF2a-ATF4, IRE1a-XBP1, and ATF6 pathway activity revealed a context-dependent increase in eIF2a-ATF4 signaling due to GSK2606414. GSK2606414 blocked eIF2a-ATF4 signaling because of the canonical ER stress agent thapsigargin. In the context of reovirus infection, GSK2606414 induced eIF2a-ATF4 signaling. Knockdown of eIF2a kinases PERK, GCN2, and PKR revealed eIF2a-ATF4 reporter activity was dependent on either PERK or GCN2. Knockdown of ATF4 abrogated the GSK2606414-induced increase in reovirus protein levels, confirming eIF2a-ATF signaling as key to the observed phenotype. Our work identifies a novel approach to enhance the efficacy and replication of reovirus in a therapeutic setting

ATR Inhibition Potentiates the Radiation-induced Inflammatory Tumor Microenvironment.

Purpose ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitize to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these drugs on the tumor microenvironment.Experimental Design: We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed posttherapy.Results Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3+ and NK cells, but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II IFN response, with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5, and CXCL10) was found in vivo, with in vitro data indicating CCL3, CCL5, and CXCL10 are produced from tumor cells after ATRi + RT.Conclusions We show that DNA damage by ATRi and RT leads to an IFN response through activation of nucleic acid-sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximize tumor control through antitumor immunity

PD-1 Blockade Following Isolated Limb Perfusion with Vaccinia Virus Prevents Local and Distant Relapse of Soft-tissue Sarcoma.

PURPOSE:The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma.Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines. RESULTS:PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines. CONCLUSIONS:Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation